Noradrenergic signaling in the central nervous system (CNS) plays an essential role in circuits involving attention, mood, appetite, memory, anxiety, and stress as well as providing pivotal support for autonomic function in the peripheral nervous system (PNS). The high affinity transporter, NET, is the primary mechanism by which noradrenergic synaptic transmission is terminated4'5. Therefore, disruption of NET regulation has the potential to impact both autonomic function and importantly mental health. In fact, cases of orthostatic intolerance have been directly linked to the expression of mutant transporters (A457P NET) which exhibit dominant negative suppression of wild type NET. Also, a recent study has demonstrated an association between attention deficit hyperactivity disorder (ADHD) and a polymorphism in the NET gene that alters promoter activity6. In addition to its fundamental role in noradrenergic signaling, NET is also a major target for psychostimulants such as cocaine and for therapeutic drugs prescribed for the treatment of depression and ADHD (e.g. amphetamine (AMPH))7. Prior studies have revealed that NET surface expression is dynamically regulated in a plethora of ways. These ways include second messenger elevation which relies on activated kinases/phosphatases, interacting proteins (e.g. syntaxinIA (SYN1A), protein phosphatase-2A (PP2Ac), protein interacts with Ckinasel (PICK1)) and both endogenous and exogenous substrates such as NE and amphetamine (AMPH), respectively. The current proposal investigates changes in NET function caused by physiological stimuli such as depolarization and upon AMPH treatment by utilizing both molecular and biochemical methods in conjunction with biophysical and real-time imaging techniques. Our preliminary data suggest that the regulation of NET function under normal conditions and during AMPH stimulation is fine-tuned by the NET N-terminus. Additionally, our data also support a role for SYN1A in both types of regulation. Therefore, our proposal is designed to enhance our understanding of NET regulation both under normal conditions and in the presence of AMPH used for the treatment of ADHD. Given the widespread influence of NE on both the CNS and PNS, and the evidence supporting a link between NET dysfunction and disease, substantial effort must be invested into enhancing our understanding of NET regulation. As our picture of NET regulation becomes more complete, so too will our ability to create unique approaches for treating disorders such as, depression, drug addiction and ADHD.